Assessment approach for evaluating high abundance protein depletion methods for cerebrospinal fluid (CSF) proteomic analysis
Assessment approach for evaluating high abundance protein depletion methods for cerebrospinal fluid (CSF) proteomic analysis
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DOI：
10.1021/pr070293w
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发表时间：
2007-09-01
影响因子：
4.4
通讯作者：
Knapp, Daniel R.
中科院分区：
文献类型：
--
作者：
Shores, Kevin S.;Knapp, Daniel R.
最佳的人脑脊液蛋白质组学分析需要去除高丰度蛋白质以便于观察低丰度蛋白质。两种免疫耗竭方法(MARS，Agilent Technologies和ProteoSeek，Piells Biotech)和一种超滤方法(50 kDa分子量截断过滤器，微孔公司)用于耗尽丰富的脑脊液蛋白质的性能进行了比较，使用图形方法以获得使用已知正常浓度范围的“标记蛋白”的分析深度。对每个耗尽的样品进行二维LC/MS/MS分析，使用MARS和ProteoSeek免疫耗竭方法分别得到171和163个独特的蛋白质鉴定，而使用50 kDa的截断分子量过滤器只鉴定了46个独特的蛋白质。鉴定的蛋白质的相对丰度用总光谱计数来估计，并与脑脊液中45种已知蛋白质的浓度进行比较，作为分析深度的标志。这项工作的结果表明，显然需要专门为去除脑脊液中的高丰度蛋白质而设计的方法学，因为与没有耗尽的分析相比，为去除高丰度血清蛋白质而设计的耗尽方法在分析深度方面几乎没有改善。在蛋白质组学分析方法的比较中，标记蛋白质方法一般可用于评估分析深度。
Optimal proteomic analysis of human cerebrospinal fluid (CSF) requires depletion of high-abundance proteins to facilitate observation of low-abundance proteins. The performance of two immunodepletion (MARS, Agilent Technologies and ProteoSeek, Pierce Biotechnology) and one ultrafiltration (50 kDa molecular weight cutoff filter, Millipore Corporation) methods for depletion of abundant CSF proteins were compared using a graphical method to access the depth of analysis using "marker proteins" with known normal concentration ranges. Two-dimensional LC/MS/MS analysis of each depleted sample yielded 171 and 163 unique protein identifications using the MARS and ProteoSeek immunodepletion methods, respectively, while only 46 unique proteins were identified using a 50 kDa molecular weight cutoff filter. The relative abundance of the identified proteins was estimated using total spectrum counting and compared to the concentrations of 45 known proteins in CSF as markers of the analysis depth. Results of this work suggest a clear need for methodology designed specifically for depletion of high-abundance proteins in CSF, as depletion methods designed to deplete high-abundance serum proteins showed little improvement in analysis depth compared to analysis without depletion. The marker protein method should be generally useful for assessing depth of analysis in the comparison of proteomic analysis methods.